Angiogenesis is the generation of new blood vessels in a tissue or organ (Carmeliet, 2005). Under normal physiological conditions, humans and animals undergo angiogenesis only in very specific and restricted situations. For example, angiogenesis is normally observed in wound healing, fetal and embryonic development, and formation of the corpus luteum, endometrium and placenta.
Angiogenesis is controlled through a highly regulated system of angiogenic stimulators and inhibitors. The control of angiogenesis has been found to be altered in certain disease states and, in many cases, the underlying pathology associated with the diseases is related to uncontrolled angiogenesis.
Both controlled and uncontrolled angiogenesis are thought to proceed in a similar manner. Endothelial cells and pericytes, surrounded by a basement membrane, form capillary blood vessels. Angiogenesis begins with the local dissolution of the basement membrane by enzymes released by endothelial cells and leukocytes. Endothelial cells, lining the lumen of blood vessels, then protrude through the basement membrane. Angiogenic stimuli promote endothelial cell migration through the eroded basement membrane. The migrating cells form a “sprout” off the parent blood vessel where the endothelial cells undergo mitosis and proliferate. The endothelial sprouts merge with each other to form capillary loops, creating a new blood vessel.
Persistent, upregulated angiogenesis occurs in many disease states, including tumor growth metastases. The diverse pathological diseases states in which upregulated angiogenesis is present have been grouped together as angiogenic-diseases, angiogenesis-associated or angiogenesis-related diseases.
One example of diseases dependent on angiogenesis is ocular neovascular diseases (Gariano and Gardner, 2005). These diseases are characterized by invasion of new blood vessels into the structure of the eye, such as the retina or cornea. They are the most common cause of blindness and comprise approximately twenty eye diseases. In age-related macular degeneration, the associated visual problems are caused by an ingrowth of choroidal capillaries through defects in Bruch's membrane, with proliferation of fibrovascular tissue beneath the retinal pigment epithelium. Angiogenic damage is also associated with diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascular glaucoma, and retrolental fibroplasias. Other diseases associated with corneal neovascularization include, but are not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, and pterygium keratitis sicca.
Another example of angiogenesis-related disease is rheumatoid arthritis (Bainbridge et al., 2006). The blood vessels in the synovial lining of the joints undergo angiogenesis. In addition to forming new vascular networks, the endothelial cells release factors and reactive oxygen species that lead to pannus growth and cartilage destruction. Angiogenesis may also play a role in osteoarthritis. The activation of the chondrocytes by angiogenic-related factors contributes to the destruction of the joint. At a later stage, the angiogenic factors promote new bone growth. Therapeutic intervention that prevents the cartilage destruction could halt the progress of the diseases and provide relief for persons suffering from arthritis.
Chronic inflammation may also involve pathological angiogenesis. Such disease as ulcerative colitis and Crohn's disease show histological changes with the ingrowth of new blood vessels into inflamed tissue. Bartonellosis, a bacterial infection found in South America, in a chronic stage is characterized by proliferation of vascular endothelial cells.
Several lines of direct evidence now suggest that angiogenesis is essential for the growth and persistence of solid tumors and their metastases. To stimulate angiogenesis, tumors upregulate the production of a variety of angiogenic factors, including the basic fibroblast growth factor (bFGF) and vascular endothelial cell growth factor (VEGF) (Yancopoulos et al., 2000). However, many malignant tumors also generate inhibitors of angiogenesis, including angiostatin, endostastin, and thrombospondin (Nyberg et al., 2005). It is postulated that the angiogenic phenotype is the result of a net balance between these positive and negative regulators of neovascularization. Several other endogenous inhibitors of angiogenesis have been identified, although not all are associated with the presence of a tumor. These include, platelet factor 4, interferon-alpha, interferon-inducible protein 10, which is induced by interleukin-12 and/or interferon-gamma, the 16 kDa N-terminal fragment of prolactin, tumstatin, arresten, canesten, anastellin, vasostatin, and vasohibin.
Neovascularization is undoubtedly a common feature of the pathology of human atherosclerotic lesions and often is found in experimental large animals (primate, pig, and dog) models of atherosclerosis and intimal thickening (Khurana et al., 2005). The strongest experimental evidence that angiogenesis plays a causative role in atherosclerosis has come from studies in the hypercholesterolemic apolipoprotein E-deficient (ApoE−/−) mouse model (Moulton et al., 1999; 2003) In this model, endostatin, angiostatin and TNP-470, three endothelium-specific inhibitors of angiogenesis, caused a remarkable reduction of plaque area. This provides the first direct evidence that angiogenesis is involved in the process of plaque formation. Increased neovascularization has also been observed at sites of intimal hyperplasia in models of arterial stenting, angioplasty, and venous bypass graft failure.
Thrombin is a serine protease, which plays a pivotal role in haemostasis. It acts as procoagulant converting fibrinogen into fibrin that anchors platelets at the site of lesion and stabilizes the clot by activating factor XIII and enhances its own generation from prothrombin by activation of factors V, VIII and XI. On the other hand, thrombin acts as an anti-coagulant by activating protein C (Di Cera, 2003).
Apart from its role in blood clotting and fibrin generation, thrombin has important roles in the initiation of angiogenesis (Tsopanoglou and Maragoudakis, 2004) Thrombin's angiogenic activity is mostly independent of its coagulant activity and is more dependent on signaling via the protease-activated receptors-1 (PAR-1). This supported by the observations obtained in mouse models, wherein a lack of thrombin generation (TF−/−, FX−/−, FV−/−, FII−/−) results in severe vascular defects in embryonic development (Moser and Patterson, 2003). Similar phenotypes occur in models of impaired thrombin binding to its PAR receptor (PAR-1−/−).
Protease-activated receptors (PARs) consists a family of G protein-coupled receptors which can be activated by proteolytic cleavage of their N-terminal extracellular domain (Ossovskaya and Bunnett, 2004). PAR-1 is the first member of this family to be cloned in which the extracellular amino terminus is cleaved to expose a new amino terminus that is involved in receptor activation (Vu et al., 1991). Subsequently, three other members of this receptor family have been identified, designated as PAR-2, PAR-3 and PAR-4. Proteolytic cleavage at the R41/S42 bond of human PAR-1 by thrombin releases a 41 amino acid peptide and unveils a tethered peptide ligand with the recognition sequence SFLLRN (SEQ ID NO:9). This sequence binds to conserved regions in the second extracellular loop of the cleaved receptor, resulting in the initiation of signal transduction. There is evidence that not only thrombin but also other molecules, such as plasmin, factor Xa, activated protein C, as well as matrix metalloprotease-1, might be able to activate this receptor under certain conditions and induce down-stream signals (Leger et al., 2006).
Thrombin, through PAR-1 signaling, interacts and stimulates a variety of vascular cells including, but is not limited to, platelets, endothelial cells, smooth muscle cells and regulates the release, expression and activation of the majority of angiogenesis mediators. For example, thrombin-induced angiogenesis in a chick chorioallontoic membrane system is associated with up-regulation of VEGF as well as angiopoietin-2 (Ang-2) (Caunt et al, 2003). Also, in endothelial cells thrombin up-regulates VEGF (Huang et al, 2001), Ang-2 (Huang et al., 2002) and the major VEGF receptor KDR (Tsopanoglou and Maragoudakis, 1999), and activates metalloproteinase-2 (Zucher et al., 1995). It was recently shown that thrombin markedly up-regulates growth-regulated oncogene-α and this chemokine in turn mediates the thrombin-induced increase of vascular regulatory proteins (MMP-1, MMP-2), growth factors (VEGF, Ang-2), and receptors (KDR) (Caunt et al, 2006). In addition thrombin induces the secretion of VEGF (Mohle et al., 1997) and Ang-1 (Li et al., 2001) from platelets. Furthermore, it was demonstrated that thrombin regulates in an opposing fashion the release of VEGF and endostatin (the potent endogenous inhibitor of angiogenesis) in platelets (Ma et al., 2005). Thrombin has also been shown to activate the proliferation of endothelial cells by acting directly as mitogenic factor (Olivot et al., 2001).
The fact that thrombin plays an important role in angiogenesis, suggests a crucial role for thrombin and its receptor, PAR-1 in tumor progression and metastasis (Nierodzik and Karpatkin, 2006). Thrombin, through PAR-1 signaling, contributes to a more malignant phenotype by activating platelet-tumor aggregation, tumor adhesion to subendothelial matrix, tumor growth and metastasis.
In addition, PAR-1 expressed on platelets and the vascular endothelium, has been shown to play important roles in normal blood vessel biology (Coughlin, 2005) and to contribute to the pathogenesis of several cardiovascular diseases including atherosclerosis, restenosis and thrombosis (Leger et al., 2006). In particular, aberrant over-expression of PAR-1 has been documented in the endothelium and vascular smooth muscle cells of human atheroscrerotic arteries, including regions of intimal thickening (Nelken et al., 1992). Activation of PAR-1 triggers mitogenic responses in smooth muscle cells and fibroblast and angiogenesis. Targeting PAR-1 with a blocking antibody reduced intimal hyperplasia by approximately 50% in a catheter-induced injury model of restenosis (Takada et al., 1998). PAR-1 deficiency also reduced restenosis in arterial injury models (Cheung et al., 1999).
Limiting infarct size by timely reperfusion is critical to improve outcomes in patients with myocardial infarction. Paradoxically, reperfusion may increase infarct size; a phenomenon known as ischemia/reperfusion injury (Ferdinandy et al., 2007). Recently, it has been shown that thrombin contributes to ischemia/reperfusion injury independently of its effects on platelets and fibrinogen. In addition, PAR-1 inhibition has been demonstrated to protect against myocardial ischemia/reperfusion injury by recruiting cardioprotective pathways (Strande et al, 2007).
Despite the wealth of information relating to the role of thrombin and PAR-1 in physiology and diseases states, the information regarding the biological role of cleaved peptides upon activation of PAR-1 is limited. There are three reports which presented evidence that correlate the 41 amino acid cleaved peptide of the PAR-1 with some platelet functions (Furman et al., 1998; 2000; 2003).